# US 4,394,444 A · Cofactor indicator compositions > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 4,394,444 A - Document ID: US4394444A - Status: Granted - Kind: A - Application number: 06/390,252 - Filed: 1982-06-21 - Granted: 1983-07-19 - Earliest claimed: 1982-06-21 - Assignees: Miles Laboratories, Inc. - Inventors: Erma C. Cameron; Claude R. Gunter; Rodric H. White-Stevens - Web page: https://patentgrep.com/patent/US4394444A ## CPC - C12Q 1/60: involving cholesterol - C12Q 1/008: {for determining co-enzymes or co-factors, e.g. NAD, ATP} - C12Q 1/26: involving oxidoreductase - C12Q 1/54: involving glucose or galactose - Y10S 435/805: Test papers - Y10S 435/81: Packaged device or kit - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS ## Abstract A composition, test device, method of making a test device and process for determination of an analyte in a liquid sample are disclosed. More particularly, there is provided a composition for the determination of an analyte in a sample which composition comprises an analyte-responsive component comprising a pyridine nucleotide susceptible of reduction in response to the presence of said analyte and at least one constituent interreactive with said analyte to cause reduction of the pyridine nucleotide, an uncoupler effective to generate an oxidizing substance from the reduced form of said pyridine nucleotide, a peroxidatively active substance, and an indicator which, when oxidized, is not susceptible to reduction by said reduced pyridine nucleotide. The pyridine nucleotide can be nicotine adenine dinucleotide (NAD) or nicotine adenine dinucleotide phosphate (NADP). ## Claims ### Claim 1 A composition for the determination of an analyte in a sample which composition comprises: an analyte-responsive component comprising a pyridine nucleotide susceptible of reduction in response to the presence of said analyte and at least one constituent interreactive with said analyte to cause reduction of the pyridine nucleotide; a hydroxylase capable of being uncoupled and a pseudosubstrate capable of uncoupling said hydroxylase, which hydroxylase and pseudosubstrate, in the presence of the reduced form of said pyridine nucleotide, are effective to generate hydrogen peroxide; a peroxidatively active substance; and a hydrazone indicator comprising a hydrazone and a coupler, which indicator, when in its oxidized form, cannot be reduced by said pyridine nucleotide. ### Claim 2 (depends on 1) The composition of claim 1 wherein said pyridine nucleotide is nicotine adenine dinucleotide. ### Claim 3 (depends on 1) The composition of claim 1 wherein said pyridine nucleotide is nicotine adenine dinucleotide phosphate. ### Claim 4 (depends on 1) The composition of claim 1 wherein said at least one interreactive constitutent is an analyte-specific enzyme. ### Claim 5 (depends on 4) The composition of claim 4 wherein said enzyme is a dehydrogenase. ### Claim 6 (depends on 5) The composition of claim 5 wherein said enzyme is lactate dehydrogenase. ### Claim 7 (depends on 5) The composition of claim 5 wherein said enzyme is .beta.-hydroxybutyrate dehydrogenase. ### Claim 8 (depends on 5) The composition of claim 5 wherein said enzyme is .alpha.-hydroxybutyrate dehydrogenase. ### Claim 9 (depends on 5) The composition of claim 5 wherein said enzyme is alcohol dehydrogenase. ### Claim 10 (depends on 5) The composition of claim 5 wherein said enzyme is a steroid dehydrogenase. ### Claim 11 (depends on 10) The composition of claim 10 wherein said enzyme is cholesterol dehydrogenase. ### Claim 12 (depends on 5) The composition of claim 5 wherein said enzyme is glucose dehydrogenase. ### Claim 13 (depends on 1) The composition of claim 1 wherein said at least one interreactive constituent is an analyte-specific enzyme and an enzyme interreactive with the product of the reaction of said analyte-specific enzyme with the analyte. ### Claim 14 (depends on 13) The composition of claim 13 wherein said analyte-responsive enzyme is a lipase and said enzyme interreactive with the product of the reaction of said lipase with the analyte is glycerol dehydrogenase. ### Claim 15 (depends on 13) The composition of claim 13 wherein said analyte-specific enzyme is hexokinase and said enzyme interreactive with the product of the reaction of said hexokinase with the analyte is glucose-6-phosphate dehydrogenase. ### Claim 16 (depends on 1) The composition of claim 1 wherein said interreactive component is a substrate of the analyte. ### Claim 17 (depends on 16) The composition of claim 16 wherein said interreactive component is a reduced substrate. ### Claim 18 (depends on 17) The composition of claim 17 wherein said interreactive component is lactate. ### Claim 19 (depends on 17) The composition of claim 17 wherein said interreactive component is .beta.-hydroxybutyric acid. ### Claim 20 (depends on 17) The composition of claim 17 wherein said interreactive component is .alpha.-hydroxybutyric acid. ### Claim 21 (depends on 17) The composition of claim 17 wherein said interreactive component is ethanol. ### Claim 22 (depends on 17) The composition of claim 17 wherein said interreactive component is a steroid. ### Claim 23 (depends on 22) The composition of claim 22 wherein said interreactive component is cholesterol. ### Claim 24 (depends on 17) The composition of claim 17 wherein said interreactive component is glucose. ### Claim 25 (depends on 1) The composition of claim 1 wherein said at least one interreactive constituent is an analyte-specific substrate and an enzyme interreactive with the product of the reaction of said analyte-specific substrate with the analyte. ### Claim 26 (depends on 25) The composition of claim 25 wherein said analyte-specific substrate is a triglyceride and said enzyme interreactive with the product of the reaction of said triglyceride with the analyte is glycerol dehydrogenase. ### Claim 27 (depends on 25) The composition of claim 25 wherein said analyte-specific substrate is glucose and said enzyme interreactive with the product of the reaction of said glucose with the analyte is glucose-6-phosphate dehydrogenase. ### Claim 28 (depends on 1) The composition of claim 1 wherein said hydroxylase capable of being uncoupled is salicylate hydroxylase and said pseudosubstrate is a benzoate. ### Claim 29 (depends on 1) The composition of claim 1 wherein said hydroxylase capable of being uncoupled is p-hydroxybenzoate hydroxylase and said pseudosubstrate is 6-hydroxynicotinate. ### Claim 30 (depends on 1) The composition of claim 1 wherein said peroxidatively active substance is a peroxidase. ### Claim 31 (depends on 1) The composition of claim 1 wherein said hydrazone is 3-methyl-2-benzothiazolinone hydrazone. ### Claim 32 (depends on 31) The composition of claim 31 wherein said coupler is primaquine. ### Claim 33 (depends on 1) A device for the determination of an analyte in a sample which comprises a carrier incorporated with the composition of claim 1. ### Claim 34 (depends on 33) A method for the determination of an analyte in a sample which comprises contacting said sample with the device of claim 33 and observing any resultant color change thereon. ### Claim 35 (depends on 1) A process for preparing a device for the determination of an analyte in a sample which comprises incorporating a carrier with the composition of claim 1. ### Claim 36 (depends on 1) A method for the determination of an analyte in a sample which comprises contacting said sample with the composition of claim 1 and observing any resultant color change.